residue, as well as two nearby positively charged (cationic) amino acids that anchor the phosphate anion in place through ionic bonding to the negatively charged phosphate. The enzyme has to configure itself so as to make room for PEP to bind to it.
Glyphosate should fit comfortably into the space reserved for the phosphate of PEP, because its extra methylphosphonate unit is very similar to phosphate, both in terms of size, shape and electric charge. It will also be attracted to the site because of the support from the nearby cationic amino acids. But, its bulky methylphosphonate unit will displace the phosphate of PEP and disrupt enzyme function in a major way.
As discussed in detail in the above referenced papers, it has been shown experimentally that a change in the DNA code, such that alanine is substituted for the glycine residue in EPSP synthase, results in a version of the protein that is completely insensitive to glyphosate [23, 24]. In a study conducted by researchers from DowDuPont, CRISPR technology was used to engineer a modified form of the native EPSP synthase enzyme of corn that showed resistance to glyphosate [25]. The first step they took was to replace the highly conserved glycine residue at the active site for PEP binding with alanine. This slightly disturbed binding to PEP completely anihilated glyphosate’s ability to suppress enzyme activity. This result can easily be explained if glyphosate’s mechanism is through direct substitution for the glycine residue in the protein’s peptide chain.
Gunatilake et al. argued that one can predict a “glyphosate susceptible motif” that consists of a highly conserved glycine residue with nearby cationic (positively charged) amino acids, such as arginine, histidine, and lysine, particularly if these amino acids coordinate at a site where a negatively charged anion binds, such as the phosphate anion in GTP or the sulfate anion in heparan sulfate [22]. This does not bode well for tTG.
5 Glyphosate and tTG
If I am right, then glyphosate substitution for glycine in tTG can be predicted to disturb its ability to bind both GTP and heparan sulfate. This will disrupt the process that keeps the enzyme in an inactive state, both inside and outside the cell, and can be predicted to have devastating consequences.
In the case of GTP, Arginine-476 and Arginine-478 supply positive charge to anchor the γ-phosphate of GTP in place [26]. These two arginine residues are in the sequence RIRVGQ (R stands for arginine and G stands for glycine).
A glyphosate residue at 480 can slip its methylphosphonate into the pocket reserved for the phosphate anion. With binding to GTP disrupted, internal molecules of tTG will remain active, able to build cross-links in multiple cytoplasmic proteins, including undigested gliadin peptides, as well as IκBα, enabling NF-κB activation, even in the absence of calcium mobilization.
Glyphosate is also likely to disrupt heparan-sulfate binding by tTG, thus allowing externally expressed tTG to cause trouble. A study designed to roughly characterize features of heparan-sulfate-binding sites in general determined that glycine, as well as the cationic amino acids lysine and arginine, were over-represented at these sites [27].
This can be expected since sulfate and phosphate are similarly sized and both negatively charged. Hence, the exact same principle that applies to phosphate-binding sites should also apply to heparan-sulfate-binding sites, making such sites likely highly sensitive to glyphosate substitution.